Abstract

Accurate motor performance depends on the integration in spinal microcircuits of sensory feedback information. Hand grasp is a skilled motor behavior known to require cutaneous sensory feedback, but spinal microcircuits that process and relay this feedback to the motor system have not been defined. We sought to define classes of spinal interneurons involved in the cutaneous control of hand grasp in mice and to show that dI3 interneurons, a class of dorsal spinal interneurons marked by the expression of Isl1, convey input from low threshold cutaneous afferents to motoneurons. Mice in which the output of dI3 interneurons has been inactivated exhibit deficits in motor tasks that rely on cutaneous afferent input. Most strikingly, the ability to maintain grip strength in response to increasing load is lost following genetic silencing of dI3 interneuron output. Thus, spinal microcircuits that integrate cutaneous feedback crucial for paw grip rely on the intermediary role of dI3 interneurons.

(A) Left: YFP+ dI3 IN with vGluT1+ boutons apposed (labeled by arrows). Right: Orthogonal sections confirming apposition of boutons labeled 1-3.(B) vGluT1+ boutons that are PV+ and PVnull on a YFP+ dI3 IN from a P7 spinal cord. Boutons in dashed boxes are magnified in Bii-Bvii. Inset in 3Bii depicts orthogonal sections of the PV+/YFP+ bouton in the Y-Z plane.(C) vGluT1+ boutons (arrowheads) on YFP+ dI3 IN from chronically transected spinal cord confirm that they do not originate from supraspinal descending inputs.All images from Isl1-YFP mice.

(A) Three types of firing behaviors. i. Tonic firing. ii. Initial bursting. iii. Delayed firing.(B) Current-clamp recording of two dI3 INs showing response to DR stimulation (20 μA, just under 3 × T, 250 μs) during current steps of three different magnitudes. i. Cell responding with an action potential followed by a prolonged hyperpolarization. ii. Cell responding with a prolonged depolarization. Arrowheads mark the time of stimulation. Top row is a scaled version of area marked by dashed box in second row.(C) Voltage-clamp recording of L5 dI3 IN depicting an EPSC in response to DR stimulation (20 μA, just under 3 × T, 250 μs) with accompanying extracellular ventral root (L5) recording. Arrowhead marks the stimulation artifact while the arrow marks the monosynaptic ventral root response.(D) EPSCs in response to DR stimulation (20 μA, just under 3 × T) reversibly blocked by CNQX.(E) drEPSCs at different holding potentials showing reversal of EPSC at depolarized potential.(F) Demonstration of monosynaptic nature of sensory input. i. Onset of drEPSC (15 μA, 3 × T) in dI3 IN as compared to onset of monosynaptic EPSC in a motoneuron from the same preparation. These two cells were recorded separately. The ventral root recording was concomitant with the motoneuron recording. The timing of the ventral root response during dI3 IN recording was the same. Blue dashed line marks the onset of the motoneuron EPSC. Red dashed line marks the onset of the dI3 IN EPSC. The difference was below 0.2 ms. ii. Top: drEPSCs (15 μA, 3 × T) in dI3 IN with low jitter (0.002 ms2). Jitter calculated on 20 responses. Bottom: drEPSCs (20 μA, 4 × T) in dI3 IN with high-jitter (0.47 ms2). iii. Low-latency, low-jitter drEPSCs in dI3 IN with no failures in response to 0.25 and 2.5 Hz stimulation frequency (n = 3), confirming these are monosynaptic responses.(G) Shift towards monosynaptic sensory inputs with age. Relation between i. Onset of drEPSC in dI3 INs and age, ii.Variance of drEPSC onset and age, and iii.Variance of drEPSC onset and onset of drEPSC.(H) Response to different strengths of DR stimulation. i. dI3 IN with increases in drEPSC magnitude with shift from low to medium strength but not to high threshold dorsal root stimulation. ii. dI3 IN with increases in drEPSC magnitude with shift from low to medium to high threshold dorsal root stimulation. T refers to the threshold at which a monosynaptic ventral root reflex was elicited. iii. Distribution of sets of responses in dI3 INs to DR stimulation (single 250 μs pulses). Low threshold fibers were considered to be recruited by stimulation between 1-2 × T, medium threshold fibers were recruited by 2-5 × T, and high threshold fibers were recruited by stimulation above 5 × T. T is the earliest stimulation strength at which a monosynaptic ventral root reflex was elicited in ventral roots. Number of cells in parentheses.(I) Diagram representing dI3 INs as part of a disynaptic pathway between sensory input and motoneurons.See also .

Reduced performance in several motor tasks involving cutaneous afferents from the paw, including loss of functional grip in dI3OFF mice

(A) During ladder walking with rung spacing of 2 cm, forelimb errors are similar in controls and mutants, whereas the mutants have more hindlimb errors than controls. Error bars represent SD.(B) Comparison of hindlimb grip of a metal bar between control and dI3OFF mice.(C) In the wire hang test, control animals grip onto the cage top, while upside down, for close to one minute whereas the dI3OFF mice are unable to hang onto the cage top while inverted. Diagram depicting minimal angle from horizontal axis at which dI3OFF mice are unable to hang onto the cage top. The grey cone represents the pooled SD.(D) Performance of control and dI3OFF mice during the wire hang test. The maximal duration of the test was one minute. Every control animals would hang on for periods longer than a minute in at least one of three trials. Similar results were observed when mice were tested a second time one or two days later. Error bars represent SD. See also and .(E) Forepaw grasp reflex in control and dI3OFFpostnatal (P1-P7) mice. Chi-square test indicated.(F) Diagram representing dI3 INs as part of a disynaptic pathway between low threshold mechanoreceptors from the skin and motoneurons involved with regulating grip strength.